Two-step immunization procedure against chlamydia infection

ABSTRACT

A host is immunized against infection by a strain of Chlamydia by initial administration of an attenuated bacteria harbouring a nucleic acid encoding a Chlamydia protein followed by administration of a Chlamydia protein in ISCOMs. This procedure enables a high level of protection to be achieved.

FIELD OF INVENTION

[0001] The present invention relates to the field of immunology and, inparticular, to a vaccination procedure for protection of a host againstdisease caused by infection with a bacterium of the Chlamydiaceasegenus, particularly Chlamydia trachomatis.

BACKGROUND OF INVENTION

[0002]Chlamydia trachomatis is a species of the genus Chlamydiacease,order Chlamydiales, C. trachomatis infects the epithelia of theconjunctivae and the genital tract, causing trachoma and a variety ofsexually transmitted diseases (STDs) which can lead to, respectively,blindness or infertility. There are at least 15 serovars of C.trachomatis, of which A, B and C are causative agents of trachoma, whileserovars D, E. F. G, H, I, J and K are the most common causative agentsof the Chlamydial STDs. C trachomatis infections are endemic throughoutthe world. Trachoma is the leading cause of preventable blindness indeveloping nations, and it is estimated that 600 million people sufferfrom trachoma worldwide, with as many as 10 million of them beingblinded by the disease. In the United States, there are an estimated 3million cases per year of STDs caused by C. trachomatis.

[0003] The pathogenesis of trachoma involves repeated ocular infectionsand the generation of a deleterious hypersensitivity response tochlamydial antigen(s) (refs. 1 to 4 —Throughout this specification,various references are referred to in parenthesis to more fully describethe state of the art of which this invention pertains. Fullbibliographic information for each citation is found at the end of thespecification. The disclosure of these references are herebyincorporated by reference into the present disclosure). The availableevidence supports the hypotheses that both secretory IgA andcell-mediated immune responses are important components of protection.Ocular infection in a primate model induces rapid and persistentproduction of IgA in tears, whereas the presence of IgG in tears istransient, corresponding to the period of peak conjunctival inflammation(refs. 5). Protective immunity following experimental ocular infectionin a sub-human primate model is homotypic and resistance to ocularchallenge correlates with the presence of serovar-specific antibodies intears (refs. 1, 2, 6). Tears from infected humans neutralized theinfectivity of homologous but not heterologous trachoma serovars for owlmonkeys eyes (ref. 7) whereas passive humoral immunization withantitrachoma antibodies was not protective (ref. 8). Several lines ofevidence indicate the importance of cell-mediated responses inprotection from or clearance of chlamydial infection. B-cell deficientmice can resolve infection, whereas nude mice become persistentlyinfected. Adoptive transfer of at least some chlamydia-specific T-celllines or clones can cure persistently infected nude mice, and thisanti-chlamydial activity is probably a function of the ability of theT-cells to secrete interferon-γ (refs. 9 to 16).

[0004] Past attempts to develop whole-cell vaccines against trachomahave actually potentiated disease by sensitizing vaccinees (refs. 1, 2).Sensitization has been determined to be elicited to a 57 kD stressresponse protein (SRP)(HSP60) present in all serovars of C. trachomatis.Repeated exposure to the 57 kD SRP can result in a delayedhypersensitivity reaction, causing the chronic inflammation commonlyassociated with Chlamydial infections. Thus, an immunogenic preparationcapable of inducing a strong and enduring mucosal neutralizing. antibodyresponse and a strong cellular immune response without sensitizing thehost would be useful (ref. 17).

[0005] A most promising candidate antigen for the development of avaccine is the chlamydial major outer membrane protein (MOMP) (refs. 18to 20). Other surface proteins and the surface lipopolysaccharide arealso immunogenic, but the antibodies they induce have not been found tobe protective (refs. 21, 33). The MOMP, which is the predominant surfaceprotein, is an integral membrane protein with a mass of about 40 kDawhich, with the exception of four variable domains (VDs) designated I,II, III and IV, is highly conserved amongst serovars. The sequences ofall four VDs have been determined for fifteen serovars (refs. 23, 24).Antibodies capable of neutralizing chlamydial infectivity recognize theMOMP (refs. 25, 26, 27, 28). Epitopes to which MOMP-specificneutralizing monoclonal antibodies bind have been mapped for severalserovars (refs. 21, 22, 29, 30, 31, 32, 33), and represent importanttargets for the development of synthetic or subunit vaccines. Thebinding sites are contiguous sequences of six to eight amino acidslocated within VDs I or II, and IV, depending on the serovar. Subunitimmunogens (e.g. isolated MOMP or synthetic peptides) containing MOMPepitopes can induce antibodies capable of recognizing intact chlamydiae(ref. 25). However, conventionally administered subunit immunogens aregenerally poor inducers of mucosal immunity. It would be useful toformulate chlamydial antigens in such a way as to enhance theirimmunogenicity and to elicit both humoral and cell-mediated immuneresponses.

[0006] Immune stimulating complexes (ISCOMs) are cage-like structuresformed from a mixture of saponins (or saponin derivatives), cholesteroland unsaturated fatty acids. The components of ISCOMs are held togetherby hydrophobic interactions, and consequently proteins which arenaturally hydrophobic (such as MOMP) or which have been treated toexpose or add hydrophobic residues can be efficiently incorporated intothe ISCOMs as they form (refs. 34, 35, 36).

[0007]C. trachomatis naturally infects the mucosal surfaces of the eyeand genital tract. Local antibody and local cellular immune responsesare an important component of protection from mucosal infections.Consequently, it would be useful for a chlamydial vaccine to induce amucosal immune response including both cellular and antibody components.

[0008] DNA immunization is an approach for generating protectiveimmunity against infectious diseases (ref. 37). Unlike protein orpeptide based subunit vaccines, DNA immunization provides protectiveimmunity through expression of foreign proteins by host cells, thusallowing the presentation of antigen to the immune system in a mannermore analogous to that which occurs during infection with viruses orintracellular pathogens (ref. 38). Although considerable interest hasbeen generated by this technique, successful immunity has been mostconsistently induced by DNA immunization for viral diseases (ref. 39).Results have been more variable with non-viral pathogens which mayreflect differences in the nature of the pathogens, in the immunizingantigens chosen, and in the routes of immunization (ref. 40). Furtherdevelopment of DNA vaccination will depend on elucidating the underlyingimmunological mechanisms and broadening its application to otherinfectious diseases for which existing strategies of vaccine developmenthave failed.

[0009] The use of attenuated bacteria, in particular S. typhimurium, hasrecently been reported for delivery of plasmid DNA for geneticimmunization (refs. 41, 42). This type of delivery offers the addedbenefit of delivering the DNA to cell types that induce a specificimmune response, such as a mucosal immune response. This type ofvaccination also offers the advantages of being safe, as many safe,attenuated strains of Salmonella are readily available, and costeffective.

[0010] EP 0192033 B1 and U.S. Pat. 5,770,714 describe the provision of aDNA construct for the expression, in vitro, of Chlamydia trachomatisMOMP polypeptides comprising the following operably linked elements:

[0011] a transcriptional promoter,

[0012] a DNA molecule encoding a C. trachomatis MOMP polypeptidecomprising a MOMP polynucleotide at least 27 base pairs in length from asequence provided in Appendix A thereto, and

[0013] a transcriptional terminator, wherein at least one of thetranscriptional regulatory elements is not derived from Chlamydiatrachomatis. There is no disclosure or suggestion in this prior art toeffect DNA immunization with any such constructs.

[0014] Copending U.S. Pat. application Ser. No. 08/893,381 filed Jul.11, 1996 (WO 98/02546), assigned to University of Manitoba and thedisclosure of which is incorporated herein by reference, describes animmunogenic composition for in vivo administration to a host for thegeneration in the host of a protective immune response to a major outermembrane protein (MOMP) of a strain of Chlamydia, comprising anon-replicating vector comprising a nucleotide sequence encoding a MOMPor MOMP fragment that generates a MOMP specific immune response, and apromoter sequence operatively coupled to the nucleotide sequence forexpression of the MOMP or MOMP fragment in the host; and apharmaceutically-acceptable carrier therefor.

[0015] Copending U.S. patent application Ser. No. 08/713,236 filed Sep.16, 1996 (WO 98/10789), assigned to Connaught Laboratories Limited andthe disclosure of which is incorporated herein by reference, describesan immunogenic composition, comprising the major outer membrane protein(MOMP) of a strain of Chlamydia, which may be Chlamydia trachomatis, andan immunostimulating complex (ISCOM).

SUMMARY OF INVENTION

[0016] The present invention provides a novel immunization strategy toprovide protection against disease caused by infection of members of theChlamydiae family, particularly Chlamydia trachomatis and materials usedtherein. The immunization strategy provided herein leads to a strongerprotective immune response than other strategies.

[0017] According to one aspect of the invention, there is provided amethod of immunizing a host against disease caused by infection byChlamydia which comprises:

[0018] initially administering to the host an immunoeffective amount ofan attenuated bacteria harbouring a nucleic acid sequence encoding atleast one immunoprotective-inducing Chlamydia protein or fragmentthereof which generates a Chlamydia protein-specific immune response,operatively connected to a eukaryotic expression element, such as thecytomegalovirus promoter, and subsequently administering to the host animmunoeffective amount of at least one purified Chlamydia protein orfragment thereof which generates a Chlamydia protein specific immuneresponse, of the same at least one Chlamydia protein or immunogenicfragment thereof as used in the initial administration, to achieve aChlamydia specific protective immune response in the host.

[0019] The attenuated bacteria may be an attenuated strain of Salmonellaor Shigella and the nucleic acid sequence may be the MOMP gene orfragments thereof from a strain of Chlamydia, including Chlamydiatrachomatis and Chlamydia pneumoniae. The boosting protein can be theMOMP protein or immunogenic fragments thereof from a strain ofChlamydia, including Chlamydia trachomatis and Chlamydia pneumoniae.

[0020] The administration steps may be effected to mucosal surfaces,such as by intranasal administration or by an initial intranasaladministration of DNA followed by intramuscular administration ofChlamydia protein.

[0021] The immune response which is achieved in the host by the methodof the invention preferably includes the production ofChlamydia-specific protection against live Chlamydia challenge andenhanced immunogenicity with greater delayed-type hypersensitivity (DTH)responses and high IgG₂ and IgG₁ antibody responses than achieved inother immunization procedures.

[0022] In another aspect, the present invention includes an attenuatedstrain of a bacterium harbouring a nucleic acid molecule encoding atleast one immunoprotection-inducing Chlamydia protein or a fragmentthereof which generates a Chlamydia protein specific immune response.The bacterium preferably is a strain of Salmonella, such as a strain ofSalmonella typhimurium. The invention extends to such attenuated strainof a bacterium when used as an immunogen and to the use of suchattenuated strain in the manufacture of an immunogen for administrationto a host.

[0023] The present invention, in a further aspect, provides a method ofimmunizing a host against infection caused by a strain of Chlamydia,which comprises:

[0024] administering to the host an immunoeffective amount of anattenuated bacteria harbouring a nucleic acid molecule encoding at leastone immunoprotection-inducing Chlamydia protein or a fragment thereofwhich generates a Chlamydia protein specific immune response. Any of theembodiments described herein with respect to the priming administrationin the prime-boost immunization protocol described herein applies tothis aspect of the invention.

BRIEF DESCRIPTION OF DRAWINGS

[0025]FIG. 1, containing panels A, B, C, D, E and F, shows theprotection results of administering the MOMP-DNA either intramuscularly(panels A, B and C) or intranasally (panels D, E and F).

[0026]FIG. 2, containing panels A, B and C, shows the protection resultsfrom mice immunized with Salmonella transfected with MOMP-DNA (pcDNA3).

[0027]FIG. 3, containing panels A, B and C, shows the protection resultsfrom mice intranasally immunized with Salmonella transfected withpcDNA3, then boosted intramuscularly with MOMP embedded in ISCOM.

[0028]FIG. 4, containing panels A, B and C, shows the DTH response(panel A) and the IgG_(2a) (panel B) and IgG₁ (panel C) antibodyresponses from mice primed intranasally with the Salmonella deliveredDNA (pcDNA3) then boosted intramuscularly with the MOMP-ISCOM protein.The data represent means±SEM of log₁₀ titres of the antibody. *represents p<0.05, when compared with naive group and group immunizedwith 10⁸ CFU pMOMP-Salmonella only.

[0029]FIG. 5 shows the elements and construction of plasmid pcDNA3/MOMP,approximately 64 kb in size.

GENERAL DESCRIPTION OF THE INVENTION

[0030] The present invention relates to methods of immunizationcomprising an initial administration of a nucleic acid sequence encodingat least one Chlamydia protein or immunogenic fragment thereof,operatively connected to a eukaryotic expression element, delivered byan attenuated Salmonella and a subsequent administration of at least oneprotein or fragment thereof of the same protein of the Chlamydia. The atleast one protein may comprise a Chlamydia protein, such as MOMP and maybe formulated into an ISCOM for administration to the host. The at leastone protein may be produced recombinantly or isolated from a chlamydialpreparation.

[0031] To illustrate the present invention, plasmid DNA was constructedcontaining the MOMP gene and MOMP gene fragments from the C. trachomatismouse pneumonitis strain (MoPn), which is a natural murine pathogen,permitting experimentation to be effected in mice. Primary infection inthe model induces strong protective immunity to reinfection. For humanimmunization, a human pathogen strain is used, such as serovar C of C.trachomatis.

[0032] Any convenient plasmid vector may be used for the MOMP gene orfragment, such as pcDNA3, a eukaryotic expression vector (Invitrogen,San Diego, Calif., USA), containing a suitable promoter, such as acytomegalovirus promoter. The MOMP gene or MOMP gene fragment may beinserted in the vector in any convenient manner. The gene or genefragments may be amplified from Chlamydia trachomatis genomic DNA by PCRusing suitable primers and the PCR product cloned into the vector. TheMOMP gene-carrying plasmid may be transferred, such as byelectroporation, into E. coli for replication therein. A MOMP-carryingplasmid, pcDNA3/MOMP, of approximately 64 kb in size, is shown in FIG.5. Plasmids may be extracted from the E. coli in any convenient manner.

[0033] The plasmid containing the MOMP gene or MOMP gene fragment may beused to transform an attenuated Salmonella bacteria according tostandard protocols, such as electroporation (ref. 43).

[0034] As described above, the primary (priming) immunization may beeffected by administration of an attenuated bacterial vector, such asSalmonella, wherein the transfected DNA is not expressed in thebacterial vector. The expression of the primary DNA is effected when thebacterial vector has released the DNA into the appropriate host cells,such as macrophages or dendritic cells. After uptake of the bacterialvector by the host cells, the auxotrophic bacteria dies after a fewrounds of division due to their inability to synthesize the essentialnutrients, such as amino acids or nucleotides. The plasmid DNA then isreleased into the cytoplasm of the infected host cells and the encodedgene expressed in the host cell.

[0035] The boosting immunization may be a Chlamydia protein incorporatedinto a immunostimulatory complex (ISCOM) or a recombinantly producedChlamydia protein. The Chlamydia protein can also be an isolated nativeChlamydia protein, which is extracted from a Chlamydia extract.

[0036] It is clearly apparent to one skilled in the art, that thevarious embodiments of the present invention may have applications inthe fields of vaccination and the treatment of Chlamydia infections. Afurther non-limiting discussion of such uses is further presented below.

EXAMPLES

[0037] The above disclosure generally describes the present invention. Amore complete understanding can be obtained by reference to thefollowing specific Examples. These Examples are described solely forpurposes of illustration and are not intended to limit the scope of theinvention. Changes in form and substitution of equivalents arecontemplated as circumstances may suggest or render expedient. Althoughspecific terms have been employed herein, such terms are intended asdescriptive and not for purposes of limitation.

Example 1

[0038] This Example illustrates the preparation of a plasmid vectorcontaining the MOMP gene.

[0039] A pMOMP expression vector was made as described in theaforementioned U.S. patent application Ser. No. 08/893,381 (WO98/02546). Briefly, the MOMP gene was amplified from Chlamydiatrachomatis mouse pneumonitis (MoPn) strain genomic DNA by polymerasechain reaction (PCR) with a 5′ primer(GGGGATCCGCCACCATGCTGCCTGTGGGGAATCCT) (SEQ ID NO:1) which includes aBamHl site, a ribosomal binding site, an initiation codon and theN-terminal sequence of the mature MOMP of MoPn and a 3′ primer(GGGGCTCGAGCTATTAACGGAACTGAGC) (SEQ ID NO:2) which includes theC-terminal sequence of the MoPn MOMP, Xhol site and a stop codon. TheDNA sequence of the MOMP leader peptide gene sequence was excluded.After digestion with BamHl and Xhol, the PCR product was cloned into thepcDNA3 eukaryotic II-selectable expression vector (Invitrogen, SanDiego) with transcription under control of the human cytomegalovirusmajor intermediate early enhancer region (CMV promoter). The MOMPgene-encoding plasmid was transferred by electroporation into E. coliDH5αF which was grown in LB broth containing 100 μg/ml of ampicillin.The plasmids was extracted by Wizard™ Plus Maxiprep DNA purificationsystem (Promega, Madison). The sequence of the recombinant MOMP gene wasverified by PCR direct sequence analysis, as described (ref. 44).Purified plasmid DNA was dissolved in saline at a concentration of 1mg/ml. The DNA concentration was determined by DU-62 spectrophotometer(Beckman, Fullerton, Calif.) at 260 nm and the size of the plasmid wascompared.with DNA standards in ethidium bromide-stained agarose gel.

[0040] The MOMP gene containing plasmid, pcDNA3/MOMP, and itsconstitutive elements are shown in FIG. 5.

Example 2

[0041] This Example illustrates DNA immunization of mice.

[0042] A model of murine pneumonia induced by the C. trachomatis mousepneumonitis strain (MoPn) was used (ref. 45). Unlike most strains of C.trachomatis, which are restricted to producing infection and disease inhumans, MoPn is a natural murine pathogen. It has previously beendemonstrated that primary infection in this model induces strongprotective immunity to reinfection. In addition, clearance of infectionis related to CD4 Th1 lymphocyte responses and is dependent on MHC classII antigen presentation (ref. 45).

[0043] Three different concentrations of MOMP-DNA were compared,administered either intramuscularly or intranasally (FIG. 1). Theresults clearly show that mucosal delivery of naked MOMP-DNA isprotective and appeared more so than intramuscularly delivered MOMP-DNA.Intranasal delivery of MOMP-DNA was evaluated in multiple experiments todetermine its reproducibility. As shown in Table 1, mucosal delivery ofMOMP-DNA evoked protective immune responses but the magnitude of theprotective index was highly variable, ranging from 0.5 to 4.1 log₁₀protection in different experiments. The basis for such variability maybe due to the limited immunogenicity of naked DNA vaccination sincechallenging vaccinated animals with a higher inoculum of MoPn markedlyreduced the protective index. Naked DNA applied to a mucosal surface mayalso have a very variable fate with some being degraded by extracellularnucleases and some being taken up the somatic cells. AGAINST CHLAMYDIAINFECTION

Example 3

[0044] This Example illustrates the delivery of DNA with attenuatedSalmonella.

[0045]Salmonella typhimurium strain 22-4 is described in ref. 46. Suchstrain was transfected with pcDNA3/MOMP and pcDNA3 by electroporation.Attenuated strains of Salmonella, transfected with plasmid DNA, werecultured for 16 to 25 hours at 37° C., without shaking in Luria Broth(LB) medium containing 100 μg/ml ampicillin. Bacteria were collected bycentrifugation and resuspended in PBS. Different concentrations ofSalmonella were diluted with PBS and the same volume of 10% sodiumbicarbonate was added immediately before immunization. Groups of 5 to 10female Balb/c mice, 6 to 8 weeks of age, were deprived of water for 5 to6 hours before immunization. Approximately 10⁵ to 10¹⁰ CFU of bacteriain 100 μl were fed by feeding needles (Ejay International Inc.). Fourinoculations at 2 week intervals were administered.

[0046] As shown in FIG. 2, mice immunized with Salmonella transfectedwith MOMP-DNA had partial protection against lung challenge with MoPn.Immunization at one mucosal surface (the gut) provides protectionagainst challenge infection at a distant mucosal surface (the lung).

Example 4

[0047] This Example illustrates a DNA prime and protein boostimmunization schedule in mice.

[0048] MOMP-DNA transfected Salmonella, prepared as described in Example3, administered at 10⁸ cfu was compared to MOMP-DNA transfectedSalmonella administered at 10⁶ cfu among groups of Balb/c mice orallyimmunized at two-week intervals on four occasions. Mice immunized with10⁶ cfu had a single protein boost intramuscularly with 1 μg MoPn MOMPembedded in ISCOM (14) at the time of the fourth immunization. The ISCOMpreparation was prepared as described in aforementioned U.S. patentapplication Ser. No. 08/718,236 (W098/10789). The mice were challengedwith 5000 IFU MoPn EB intranasally two weeks after the lastimmunization. Challenged mice were sacrificed at day 10 postinfection.The body weight was measured daily after infection until mice weresacrificed (FIG. 3, panel A). These mice were much better protected thanmice given 10⁸ cfu Salmonella without a protein boost, as described inExample 3. Chlamydia EB growth in the lungs at day 10 postinfection wasanalyzed by quantitative tissue culture (FIG. 3, panel B and C). In.FIG. 3, panel B, the data represents the mean±SEM of log₁₀ IFU per lungof 5 to 6 mice and panel C represents the results observed in individualmice. DNA primed, protein boosted mice also demonstrated enhancedimmunogenicity with greater DTH responses (FIG. 4, panel A) and higherserum IgG₂ and IgG₁ antibody responses (FIG. 4, panels B and C). Serawere collected from immunized mice 2 weeks after the last immunization.MoPn-specific IgG_(2a) (panel B) and IgG₁ (panel C) antibodies weretested by ELISA.

Example 5

[0049] This Example describes the measurement of MoPn-specificdelayed-type hypersensitivity (DTH).

[0050] To evaluate DTH, 25 μl of ultraviolet (UV)-killed MoPn EBs (2×10⁵IFU) in SPG buffer 25 was injected into the right hind footpad of miceand the same volume of SPG buffer was injected into the left hindfootpad as a control. Footpad swelling was measured at 48 hours and 72hours post injection using a dila-gauge caliper. The difference betweenthe thickness of the two footpads was used as a measure of the DTHresponse.

SUMMARY OF DISCLOSURE

[0051] In summary of this disclosure, the present invention providesmethods of immunizing a host against Chlamydia infection using DNAcarried by an attenuated bacteria and materials used in such procedures.Modifications are possible within the scope of the invention. TABLE 1Intranasal (IN) immunization with MOMP-DNA evokes protective immunity toChlamydia trachomatis MoPn lung infection. PROTEC- EXPERIMENT LOG10IFU/LUNG TIVE CHALLENGE Number PcDNA3-IN pMOMP-IN Index Inoculum (IFU) 24.93 ± 0.68 3.65 ± 0.94 1.28 1000 (N = 7) (N = 6) 3  6.1 ± 0.32  3.0 ±1.15 4.1 5000 (N = 4) (N = 4) 4  4.4 ± 0.32  3.9 ± 0.13 0.5 5000 × 2 (N= 7) (N = 7) 7 5.39 ± 0.3  3.8 ± 0.63 1.59 5000 (N = 8) (N = 8)

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1 2 1 35 DNA Chlamydia trachomatis 1 ggggatccgc caccatgctg cctgtggggaatcct 35 2 28 DNA Chlamydia trachomatis 2 ggggctcgag ctattaacgg aactgagc28

What we claim is:
 1. A method of immunizing a host against infectioncaused by a strain of Chlamydia, which comprises: initiallyadministering to the host an immunoeffective amount of an attenuatedbacteria harbouring a nucleic acid molecule encoding at least oneimmunoprotection-inducing Chlamydia protein or a fragment thereof whichgenerates a Chlamydia protein specific immune response, and subsequentlyadministering to the host an immunoeffective amount of at least onepurified Chlamydia protein or a fragment thereof which generates aChlamydia protein specific immune response, of the same at least oneChlamydia protein as the initial administration to achieve a Chlamydiaspecific protective immune response in the host.
 2. The method of claim1 wherein said immunoprotection inducing Chlamydia protein or fragmentthereof is a major outer membrane protein (MOMP) of a strain ofChlamydia.
 3. The method of claim 2 wherein said strain of Chlamydia isa strain of Chlamydia pneumoniae.
 4. The method of claim 2 wherein saidstrain of Chlamydia is a strain of Chlamydia trachomatis.
 5. The methodof claim 1 wherein said nucleic acid molecule is provided in a vectorcomprising the same and a promoter sequence operatively coupled to saidnucleic acid molecule for expression of said Chlamydia protein orfragment thereof in said host.
 6. The method of claim 5 wherein saidnucleic acid molecule encodes a full-length major outer membrane protein(MOMP) of a strain of Chlamydia.
 7. The method of claim 6 wherein saidstrain of Chlamydia is a strain of Chlamydia pneumoniae.
 8. The methodof claim 6 wherein said strain of Chlamydia is a strain of Chlamydiatrachomatis.
 9. The method of claim 1 wherein said attenuated bacteriais an attenuated strain of Salmonella.
 10. The method of claim 5 whereinsaid promoter is a cytomegalovirus promoter.
 11. The method of claim 5wherein said vector is a plasmid vector.
 12. The method of claim 11wherein said plasmid vector has the identifying characteristics ofpcDNA3/MOMP as seen in FIG.
 5. 13. The method of claim 1 wherein saidimmunoprotection-inducing chlamydial protein used in said subsequentadministration step is administered incorporated into animmunostimulating complex (ISCOM).
 14. The method of claim 13 whereinsaid chlamydial protein or fragment thereof is a major outer membraneprotein (MOMP) of a strain of Chlamydia.
 15. The method of claim 14wherein said strain of Chlamydia is a strain of Chlamydia pneumoniae.16. The method of claim 14 wherein said strain of Chlamydia is a strainof Chlamydia trachomatis.
 17. The method of claim 1 wherein said firstadministration step is effected to mucosal surfaces.
 18. The method ofclaim 17 wherein said first administration step is effected byintranasal administration and said second administration step iseffected by intramuscular administration.
 19. An attenuated strain of abacterium harbouring a nucleic acid molecule encoding at least oneimmunoprotection-inducing Chlamydia protein or a fragment thereof whichgenerates a Chlamydia protein specific immune response.
 20. Theattenuated strain of claim 19 wherein said immunoprotection inducingChlamydia protein or fragment thereof is a major outer membrane protein(MOMP) of a strain of Chlamydia.
 21. The attenuated strain of claim 20wherein said strain of Chlamydia is a strain of Chlamydia pneumoniae.22. The attenuated strain of claim 20 wherein said strain of Chlamydiais a strain of Chlamydia trachomatis.
 23. The. attenuated strain ofclaim 19 wherein said nucleic acid molecule is provided in a vectorcomprising the same and a promoter sequence operatively coupled to saidnucleic acid molecule for expression of said Chlamydia protein orfragment thereof in said host.
 24. The attenuated strain of claim 23wherein said promoter is a cytomegalovirus promoter.
 25. The attenuatedstrain of claim 23 wherein said vector is a plasmid vector.
 26. Theattenuated strain of claim 25 wherein said plasmid vector has theidentifying characteristics of pcDNA3/MOMP as seen in FIG.
 5. 27. Theattenuated strain of claim 19 wherein said attenuated bacteria is anattenuated strain of Salmonella.
 28. The attenuated strain of claim 27wherein said attenuated strain of Salmonella is an attenuated strain ofSalmonella typhimurium.
 29. A method of immunizing a host againstinfection caused by a strain of Chlamydia, which comprises:administering to the host an immunoeffective amount of an attenuatedbacteria harbouring a nucleic acid molecule encoding at least oneimmunoprotection-inducing Chlamydia protein or a fragment thereof whichgenerates a Chlamydia protein specific immune response.
 30. The methodof claim 29 wherein said immunoprotection inducing Chlamydia protein orfragment thereof is a major outer membrane protein (MOMP) of a strain ofChlamydia.
 31. The method of claim 30 wherein said strain of Chlamydiais a strain of Chlamydia pneumoniae.
 32. The method of claim 30 whereinsaid strain of Chlamydia is a strain of Chlamydia trachomatis.
 33. Themethod of claim 29 wherein said nucleic acid molecule is provided in avector comprising the same and a promoter sequence operatively coupledto said nucleic acid molecule for expression of said Chlamydia proteinor fragment thereof in said host.
 34. The method of claim 33 whereinsaid promoter is a cytomegalovirus promoter.
 35. The method of claim 33wherein said vector is a plasmid vector.
 36. The method of claim 35wherein said plasmid vector has the identifying characteristics ofpcDNA3/MOMP as seen in FIG.
 5. 37. The method of claim 29 wherein saidattenuated bacteria is an attenuated strain of Salmonella.
 38. Themethod of claim 37 wherein said attenuated strain of Salmonella is anattenuated strain of Salmonella typhimurium.
 39. The method of claim 29wherein said administration is effected to mucosal surfaces.
 40. Themethod of claim 39 wherein said administration is effected by intranasaladministration.